Factors affecting high-frequency fungal protoplast fusion

Summary Factors influencing the fusion frequency of protoplasts have been examined with auxotrophic mutants ofAspergillus nidulans. The optimum conditions were a total of 5 to 15 million protoplasts per ml, and 25% polyethylene glycol (PEG) 4000 or 6000 as fusogenic agent in 10 to 100 mM CaCl₂ solution.     

Ethylene production and cell wall formation in mesophyll protoplasts

Summary The regulation of ethylene synthesis, in relation to the presence of a new cell wall, has been investigated forNicotiana sylvestris leaf protoplasts. It is clear that the production of ethylene is not controlled by the new wall, which has no action on the ethylene formation. The addition of dichlorobenzonitrile to cultivated protoplasts causes the inhibition of wall formation, without any other apparent deleterious effects.     

Reversion of Lactobacillus lactis protoplasts

More than 99% of L. lactis cells have been converted to protoplasts upon digestion of cell walls with mutanolysin (N-(acetyl)muramidase). Functional protoplasts were obtained even with the lowest level of the enzyme that was used (0.1 U.ml-1 of the cell suspension) and after incubation at 37 degrees C for 2 min. The regeneration of the polymerized cell wall appears to be induced by a cell homogenate of the same organism.     

Antikörperproduktion mit isolierter Bakterienzellwand und mit Protoplasten

Summary The cell wall of an aerobic bacillus isolated with trypsin treatment produces in rabbits a polysaccharide antibody which is responsible for the majority of the serological reactions of the cell surface.After digestion of the cell wall polysaccharide with lysozym, intact, naked protoplasts can be obtained in isotonic saccharose solution. This produces in rabbits an antiserum which contains no polysaccharide antibody and which reacts not only with the protoplasts but also with a thermolabile protein of the bacterial surface.     

Transfer of isolated nuclei into protoplasts of Aspergillus nidulans.

Nuclei were isolated from protoplasts of a haploid auxotrophic Aspergillus nidulans strain. Transformation of protoplasts prepared from a complementary haploid auxotrophic strain with these purified nuclei resulted in both heterokaryotic and diploid colonies. The nutritionally-complementing colonies appeared at a frequency of 5 x 10(-7) to 10(-8).     

The isolation,culture and organ differentiation from mesophyll protoplasts ofMollugo nudicaulis Lam., a C4 species

Summary Isolation and culture of mesophyll protoplasts fromMollugo nudicaulis, a C₄ species, is reported. Protoplasts developed into callus with root formation. However, no shoot was differentiated. This work was carried out as a part of our overall objective of inducing somatic fusion between the protoplasts of C₃ and C₄ species ofMollugo.     

Transfer of isolated nuclei into protoplasts ofAspergillus nidulans

Nuclei were isolated from protoplasts of a haploid auxotrophicAspergillus nidulans strain. Transformation of protoplasts prepared from a complementary haploid auxotrophic strain with these purified nuclei resulted in both heterokaryotic and diploid colonies. The nutritionally-complementing colonies appeared at a frequency of 5×10^–7 to 10^–8.     

Isolation and fusion studies on protoplasts from pollen tetrads.

Different enzymes were tested for isolation of intact protoplasts from pollen tetrads. About 80% isolation was achieved from pollen tetrads of Cajanus cajan and Zea mays and about 60% from Luffa cylindrica and Lycopersicon esculentum after 4 h of treatment with 5% cellulase. When these mononucleate protoplasts were incubated in presence of 0.05 M CaCl2 in 0.3 M glucose at pH 10.5, 70-80% fusion was achieved. Fusion was rare in sodium nitrate solutions.     

Menadione-induced apoptosis and the degradation of lamin-like proteins in tobacco protoplasts

Detection of stereotypic hallmarks of apoptosis during cell death induced by menadione, including DNA laddering and the formation of apoptotic bodies, is reported. Comet assay and the TdT-mediated dUTP nick end labelling (TUNEL) procedure were also performed to detect DNA fragmentation. Inhibition of DNA fragmentation by Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and phenylmethylsulfosyl (PMSF) implicated the involvement of caspase-like proteases in menadione-induced apoptosis in plants. We further studied the cleavage of lamin-like proteins during apoptosis in menadione-treated tobacco protoplasts. In animals, it has been reported that the solubilization of nuclear lamina and lamin degradation occurs during apoptotic cell death. However, little is known about the fate of lamins in apoptotic plant cells. Our study provided evidence that lamin-like proteins degraded into 35-kDa fragments in tobacco protoplasts induced by menadione, and this preceded DNA fragmentation. The results thus indicated that proteolytic cleavage of nuclear lamins was also conserved in programmed cell death in plants. Received 16 November 1998; received after revision 21 December 1998; accepted 23 December 1998     

Isolation and fusion studies on protoplasts from pollen tetrads

Summary different enzymes were tested for isolation of intact protoplasts from pollen tetrads. About 80% isolation was achieved from pollen tetrads of Cajanus cajan and Zea mays and about 60% from Luffa cylindrica and Lycopersicon esculentum after 4 h of treatment with 5% cellulase. When these mononucleate protoplasts were incubated in presence of 0.05 M CaCl₂ in 0.3 M glucose at pH 10.5, 70–80% fusion was achieved. Fusion was rare in sodium nitrate solutions.This work was supported by a grant from the Indian Council of Agricultural Research, New Delhi (No. 1-32/73, Ed. I) in the form of a Senior Fellowship awarded to P. C. D.Work carried out at Molecular Cytogenetics Research Unit, Dept. of Genetics and Plant Breeding, Banaras Hindu University, Varanasi, India.     

Damage by ozone to the mechanical integrity of the protoplast plasmalemma

Summary Ozone acts on the plasmalemma as to weaken its mechanical properties. This results in the bursting of protoplasts.This work was carried ont with the help of a grant by the FCAC, Ministère de l'Education du Québec, Canada.     

Transcription of bacterial DNA by isolated plant nuclei.

Plant nuclei prepared from protoplasts can be used as a cell-free system for testing their template activity of procaryotic DNA for plant polymerases. We were able to demonstrate that plant polymerases of Petunia hybrida are capable of transcribing linear bacterial DNA, whereas supercoiled DNA could not be used as a template.     

The effect of actinomycin D on the production of viruses by the protoplasts of Chinese cabbage infected in vitro by the yellow mosaic virus of turnips]

Chinese Cabbage (Brassica sinensis L. var. Cantonner) protoplasts were infected by Turnip yellow mosaic virus (TYMV) and inoculated in the presence or absence of actinomycin D. Virus production was determined 40 hrs. after inoculation, the time required for the virus replication cycle to be terminated. While actinomycin D had no effect on TYMV production when present at a concentration of 1 microng/ml, a 50 to 80% inhibition of virus production was noticed at concentrations of the order of 5 to 10 microng/ml, and the inhibition reached 90% with 25 microng/ml.     

The use of cell free extracts derived from fungal protoplasts in the study of aflatoxin biosynthesis.

A supernatant fraction derived from protoplasts of Aspergillus flavus was shown to be capable of converting both sterigmatocystin and versiconal hemiacetal acetate to aflatoxin B1. Versicolorin A was not converted under the same conditions.     

The use of fungal protoplasts in the study of aflatoxin biosynthesis.

Protoplasts derived from Aspergillus flavus are shown to be capable of synthesizing aflatoxins when incubated in a chemically defined medium. 14C-Acetate and 14C-Versicolorin A, added to protoplasts from 3-day-old mycelium, are incorporated into aflatoxin B1.     

The use of cell free extracts derived from fungal protoplasts in the study of aflatoxin biosynthesis

Summary A supernatant fraction derived from protoplasts ofAspergillus flavus was shown to be capable of converting both sterigmatocystin and versiconal hemiacetal acetate to aflatoxin B₁. Versicolorin A was not converted under the same conditions.     

Regeneration of plants from mesophyll protoplasts ofAtropa belladonna

Summary Isolated leaf mesophyll protoplasts ofAtropa belladonna when cultured in defined liquid culture media regenerate cell walls, divide and form calli. Subsequent induction of shoot and root organogenesis leads to plantlets which grow to maturity after transfer to soil.Acknowledgment. We are indebted to Václav Mandák for skilful technical assistance.     

The use of fungal protoplasts in the study of aflatoxin biosynthesis

Summary Protoplasts derived fromAspergillus flavus are shown to be capable of synthesizing aflatoxins when incubated in a chemically defined medium.¹⁴C-Acetate and¹⁴C-Versicolorin A, added to protoplasts from 3-day-old mycelium, are incorporated into aflatoxin B₁.     

Zur Arteinteilung der GattungStreptomyces Waksman et Henrici

Summary In the existing systems of the genusStreptomyces Waksman et Henrici many characters of doubtful or low systematic value were considered to differentiate the species. Following our experiences of many years and with many strains, we came to the conviction that a reliable species-distinction is only possible if definitely determinable and stable characters are considered. In the genusStreptomyces, such characters are (1) morphology of the spores; (2) colour of the aerial mycelium; (3) morphology of the aerial mycelium; (4) formation of a melanoid pigment.